Intracellular transport of organelles and particles is ubiquitous in animal cells and has fundamental importance for such diverse processes as secretion, neuronal signaling, organization of endomembranes, and cell division. The driving force for intracellular transport is provided by molecular motors bound to the surface of cargo organelles and moving along microtubules (MTs) and actin filaments (AFs). Because several types of motors are simultaneously bound to the same cargo and the same organelles move along both types of cytoskeletal tracks transport events have to be precisely regulated to ensure the delivery of organelles and particles to specific cellular destinations but the exact regulatory mechanisms are largely unknown. Here we propose to examine the mechanisms of regulation of intracellular transport using melanophores as an experimental system. The major function of these cells is fast and synchronous redistribution of thousands of membrane-bounded pigment granules, which aggregate at the cell center or redisperse uniformly throughout the cytoplasm by moving along MTs and AFs by means of cytoplasmic dynein (aggregation), and kinesin 2 and myosin V (dispersion). Switching between these motors and cytoskeletal systems is regulated by a single second messenger, cAMP, and involves the activity of Protein Kinase A (PKA). Background data by the PI demonstrates that PKA forms two separate complexes on the surface of pigment granules with molecular motors involved in pigment aggregation and pigment dispersion; these complexes are called "regulated motor units" (RMU). Preliminary evidence also indicates that MTs and AFs themselves may play an active role in pigment transport. In this research project biochemical, molecular, and cellular approaches will be used to test the hypothesis that multiple mechanisms are involved in the regulation of intracellular transport in melanophores. To examine these regulatory mechanisms, signaling components of RMU will be identified, and their role in the regulation of molecular motors will be elucidated. The role of the properties of cytoskeletal tracks themselves in the regulation of intracellular transport will be also determined.